Eimeria stiedae infects the epithelium of bile ducts of the liver and causes economic losses for the rabbit industry. In this study, we aimed to study the infection rate and histopathology and to characterize Eimeria stiedae from Ashmoun and Sadat City, Minoufiya governorate, Egypt by a nested polymerase chain reaction. Specific PCR and nested PCR primers to the ITS-2 gene of Eimeria stiedae were used. The infection rate was 12.5%. The infection rate among localities was 19% for Ashmoun and 7% for Sadat. The infection rate among sex groups was 12.35% for females and 13.33% for males. The infection rate among age groups was 11.11% for >1year and 15.38% for E. stiedae and its stages were observed. There was hydropic degeneration in hepatocytes. The infection significantly increased ALT, AST, ALP, GGT, total bilirubin, direct bilirubin, indirect bilirubin, and urea while decreased PCV, Hb, RBCS, MCH, MCHC, and lymphocytes. The PCR and nested PCR amplified the expected bands of the ITS-2 gene. The PCR and nested PCR products were sequenced. The sequences had high identity percent with E. stiedae isolates from China (LY) and Egypt (BSU-1). Nested PCR of the ITS-2 gene might be useful in the molecular diagnosis of E. stiedae in rabbits.